Técnicas calorimétricas aplicadas ao estudo termodinâmico das iterações entre proteínas e polissacarídeos Monique Barreto SantosI Bernardo de Sá Costa

Calorimetric techniques applied to the thermodynamic

study of interactions between proteins and polysaccharides

Técnicas calorimétricas aplicadas ao estudo termodinâmico

das iterações entre proteínas e polissacarídeos

Monique Barreto Santos

_{Bernardo de Sá Costa}

_{Edwin Elard Garcia Rojas}

ISSN 1678-4596

ABSTRACT

The interactions between biological macromolecules have been important for biotechnology, but further understanding is needed to maximize the utility of these interactions. Calorimetric techniques provide information regarding these interactions through the thermal energy that is produced or consumed during interactions. Notable techniques include differential scanning calorimetry, which

generates a thermodynamic profile from temperature scanning, and

isothermal titration calorimetry that provide the thermodynamic parameters directly related to the interaction. This review described how calorimetric techniques can be used to study interactions between proteins and polysaccharides, and provided valuable insight into the thermodynamics of their interaction.

Key words: isothermal titration calorimetry, differential scanning calorimetry, calorimetric techniques, biomolecules, proteins.

RESUMO

As interações entre macromoléculas biológicas têm tido importante aplicação na biotecnologia, mas, para sua devida utilização, estudos mais detalhados são necessários. As técnicas calorimétricas permitem estudá-las ao serem capazes de fornecer informações referentes a essas interações através da energia térmica que é gerada ou absorvida durante o processo de interação. Dentre as técnicas que mais se destacam estão a Calorimetria Exploratória Diferencial, que é capaz de fornecer um

perfil termodinâmico a partir de uma varredura de temperatura, e a Calorimetria de Titulação Isotérmica, que fornece parâmetros termodinâmicos diretamente relacionados ao processo de

interação. Nesta revisão, descrevemos como essas técnicas calorimétricas podem ser efetivamente aplicadas no estudo das interações entre proteínas e polissacarídeos, com o propósito de

obter informações valiosas sobre a termodinâmica da interação.

Palavras-chave: calorimetria de titulação isotérmica, calorimetria exploratória diferencial, técnicas calorimétricas, biomoléculas, proteínas.

INTRODUCTION

Characterizing the interactions between

macromolecules greatly enhances understanding of

biological systems and is useful for various applications

in biotechnology. Macromolecular interactions can

include bonds between substrates and enzymes,

antigens and antibodies and smaller molecules like

drugs and hormones linked to carrier proteins or

receptor (ARMSTRONG et al., 2013; NADEMI et

al., 2013; CAO et al., 2013; RÀFOLS et al., 2014). In

addition, proteins and polysaccharides are increasingly

used in technological applications to form new products

(DIARRASSOUBA et al., 2015) and understanding

how they interact will be critical to future technologies.

Almost all physical, chemical, or biological

processes result in the produce or consume of thermal

energy. Calorimetry, which means measuring heat,

is the general term that describes all experiments

in which thermal energy is measured in function

of time or temperature. (WADSÖ, 1986; IUPAC,

1994; BROWN, 1998; GAISFORD & BRUCKTON,

2001; RUSSEL, et al., 2009). Currently, the term

microcalorimetry defines heat measurements in a

– REVIEW –

I_{Programa de Pós-graduação em Ciência e Tecnologia de Alimentos (PPGCTA), Universidade Federal Rural do Rio de Janeiro (UFRRJ),}

Seropédica, RJ, Brasil.

II_{Laboratório de Engenharia e Tecnologia Agroindustrial (LETA), Universidade Federal Fluminense (UFF), Av. dos Trabalhadores, 420,}

27255-125, Volta Redonda, RJ, Brasil. E-mail: edwin@vm.uff.br. *_{Corresponding author.}

microwatt range (RUSSEL et al., 2009; IUPAC,

2014). Measuring the heat flow in the calorimeter

gives insight into the thermodynamic, chemical, and

structural properties of a molecule (BROWN, 1998).

Calorimetric techniques are advantageous

because they do not depend on the physical nature

of the sample, rarely require any prior treatment, and

are completely non-invasive. Furthermore, obtaining

continuous and real-time data is an appealing aspect

of these techniques. However, the high sensitivity and

the non-specificity have both benefits and drawbacks

because an improper sample preparations can cause

incorrect interpretations of the results (WADSÖ,

1986; GAISFORD & BRUCKTON, 2001; RUSSEL,

et al., 2009).

Calorimetric

techniques

that

are well suited for the study of macromolecular

interactions include isothermal titration calorimetry

(ITC) and differential scanning calorimetry

(DSC). ITC measures heat flow as a function of

time and DSC measures heat flow as a function of

temperature (GAISFORD & BRUCKTON, 2001).

Table 1 shows different applications that use ITC

and/or DSC to study interactions between proteins

and polysaccharides. This review article describes

each calorimetric technique and its application to

understanding of interactions between proteins and

polysaccharides in food systems.

Differential scanning calorimetry (DSC)

The International Union of Pure and

Applied Chemistry (IUPAC, 2014) defines thermal

analysis as the study of the relationship between a

property of the sample and its temperature when

it is heated or cooled in a controlled manner. DSC

is more than a calorimetric technique; it is also

considered a thermal analysis, where the physical

property being studied is heat (IUPAC, 1994). DSC

studies transitions or processes that gain or lose heat

as a function of temperature in other words, when

a substance is subjected to a temperature change,

endothermic (heat absorption) or exothermic (heat

generation) processes may occur. As most biological

molecules of interest undergo transformations when

subjected to temperature variations, it is possible to

use DSC to determine the energy involved in such

processes (JOHNSON, 2013).

In a DSC analysis, the sample and reference

are heated in a controlled way. As a result, the instrument

measures the difference of heat capacity at constant

pressure (

C

) (WADSÖ, 1986; HEERKLOTZ, 2004).

The

C

is defined as the ability of the sample to absorb

or release energy without changing temperature.

C

is a fundamental property derived from other

thermodynamic parameters such as enthalpy change

(Δ

H

) and entropy change (Δ

S

), defined as follows

(equations 1 by invoking the Kirchhoff equation)

(PIRES et al., 2009; PRIVALOV, 2015):

(1)

The thermodynamic properties may be

evaluated in accordance with the following standard

relations (equations 2 to 4) (PRIVALOV, 2015):

Δ

H

(

T

) = Δ

H

(

T

) – ΔC

(

T

–

T

) (2)

Table 1 - Application of calorimetric techniques in interactions between polysaccharides and proteins.

Biomolecular interaction Calorimetric technique Thermodynamic parameters Reference

β-lactoglobulin and Chitosan ITC n and ΔH GUSEY & MCCLEMENTS, 2006

β-lactoglobulin–sodium alginate ITC ΔH HARNSILAWAT et al., 2006

BSA and Dextran Sulfate DSC Tm and ΔH ANTONOV & WOLF, 2005

β- lactoglobulin and Dextran Sulfate DSC Tm and ΔH m VARDHANABHUTI et al., 2009

WPI and Chitosan ITC ΔH BASTOS et al., 2010

Casein and Dextran DSC Tg HERNANDEZ et al., 2011

β- lactoglobulin and Gum Arabic ITC K, ΔH, n, ΔS, ΔG e ΔCp ABERKANE et al., 2012

β- lactoglobulin and Carrageenan ITC n, K, ΔH ΔS e ΔG HOSSEINI et al., 2013.

Soy protein and Chitosan DSC Tm and ΔH m YUAN et al., 2014.

Pectin and WPI DSC Tm MAO et al., 2014.

Hyaluronic Acid and Lysozyme DSC e ITC Tm, K, ΔH, ΔS, ΔG WATER et al., 2014.

Soy protein and Gum Arabic ITC n and ΔH DONG et al., 2015.

β- lactoglobulin and Lactoferrin ITC K, ΔH, n, ΔS, ΔG TAVARES et al., 2015.

Polysaccharides (carrageenan and

CMC) and soy proteins DSC Tm and ΔH m SPADA et al., 2015

n: Stoichiometry; ΔH : Enthalpy change; Tm: Melting temperature; ΔH m : Melting enthalpy change; K: Equilibrium binding constant; ΔS:

Δ

S

(

T

) = Δ

H

(

T

) /

T

– Δ

C

ln

(

T

|

T

) (3)

Δ

G

(

T

) = Δ

H

(

T

) –

T

Δ

S

(

T

) (4)

Considering the equations above, when

Δ

H

is negative and Δ

S

is positive, the free energy

(Δ

G

) is negative and the interaction is spontaneous

(O’BRIEN et al., 2001). DSC can provide a complete

thermodynamic characterization of interactions

induced by temperature. Regarding proteins, the

literature has shown its application in determinating

the melting enthalpy change (Δ

H

) and melting

temperature (

T

) (CAO et al., 2008; DAMODARAN &

AGYARE 2013; TABILO-MUNIZAGA et al., 2014)

and regarding polysaccharide measurements, DSC

has been used to identify the glass transition (

Tg

) (LIU

et al., 2007; HOMER et al., 2014). For biomolecular

interactions, DSC can identify an increase or decrease

in the denaturation temperature and glass transition

after an interaction compared to isolated molecules

(VARDHANABHUTI et al., 2009; HERNÁNDEZ et

al., 2011; HOMER et al., 2014; MAO et al., 2014)

Most recent equipment used in DSC are

highly sensitive and also highly stable. They also have

large dynamic measurement ranges (below 0°C to over

100°C, under pressure) (PRIVALOV & DRAGAN,

2007). In general, calorimeters control minimum

temperature variation between the reference cell

containing the buffer, and the sample cell containing

the molecule of interest diluted with the buffer, both

subjected to the same temperature program. As the

changes temperature processes that generate or absorb

energy (heat) occur in the sample cell and produce a

temperature difference between the two cells. Heaters

around the cells, to keep the difference between cells

equal to zero, respond increasing the temperature in

the reference cell when the process is exothermic and

increasing the temperature of the sample cell when

the process is endothermic. The amount of energy

necessary to maintain thermal balance within the

system is proportional to the energy change occurring

in the sample (PIRES et al., 2009; JOHNSON, 2013).

There is also a heat flux DSC instrument

that has a single heating system. For this instrument,

the temperature difference between the sample and the

reference cell is logged as the direct measure of the

difference between heat flow rates (BROWN, 1998).

However, this method overall is less accurate because

the temperature measurement is less accurate than the

energy input measurements (PIRES et al., 2009). To

perform this calorimetric technique, all parameters such

as heating rate, sample concentration, pH, presence of

solutes, kind of container and instrumentation must

be well defined for results to be interpreted (MA &

HARWALKAR, 1996; HOMER et al., 2014).

Interactions between proteins and

polysaccharides and their effect on the thermal stability

of compounds have been the focus of many studies.

However, results have shown variations according to

the macromolecules used. Two studies evaluated the

influence of Dextran in thermal stability of Bovine

Serum Albumin (BSA) (ANTONOV & WOLF, 2005)

and β-Lactoglobulin (β-Lg) (VARDHANABHUTI

et al., 2009) and concluded that in both cases the

polysaccharide was able to reduce the thermal stability

of the protein in low pH and in high concentrations of

Dextran. Another study evaluated the effect of Dextran

on Casein and reported that the glass transition value

(

Tg

) of Dextran decreased while crystallization

temperature increased in the presence of casein

(HERNÁNDEZ et al., 2011). These phase transitions

must be considered in food processing and storage.

When interactions between soy protein

fractions (7S and 11S) and chitosan (CS) were studied

during temperature increases from 30 to 120°C at the rate

of 5°C/min, the measurements revealed endothermic

processes indicating the denaturation point (YUAN et

al., 2014). According to the authors, the interaction with

formation of coacervates substantially increased

ΔH

and

T

compared to the protein alone. This suggested

that the coacervation between soy protein fractions and

chitosan increased the thermal stability of the protein.

In contrast, a different study on the thermal behavior of

whey protein isolates (WPI) in the absence or presence

of pectin reported that molecular interactions reduced

the stability of WPI (MAO et al., 2014).

Isothermal titration calorimetry (ITC)

ITC measures the energy released during

molecular interactions and is used for the qualitative

and quantitative characterization of them (HAPPI

EMAGA, et al., 2012; OGNJENOVIĆ et al., 2014).

A typical interaction system involves the vacant

binding site, the free ligand, and the complex at some

equilibrium in solution. Understanding the interaction

requires knowing the equilibrium constant for the

binding process (

K

) and the binding stoichiometry (

n

).

Equations 5 to 8 illustrate the relevant thermodynamic

relationships (FREREY & LEWIS, 2008).

(5)

Δ

G

₌

_–

_RT

_ln

_K

eq

(6)

(7)

Δ

G =

Δ

H

–

T

ΔS (8)

Where Δ

G

_{is the standard Gibbs free}

Among the techniques that evaluate

thermodynamic interactions, only ITC provides a

several thermodynamic parameters (

K

,

n

, Δ

G

, Δ

H

, and

Δ

S

) in a single titration. It requires only small amounts

of sample and does not need molecular marker which

may generate interference (FREYER & LEWIS, 2008;

RAJARATHNAM & RÖSGEN, 2014). Δ

H

is related

to the energy involved in molecular interactions

and reflects the contribution of hydrogen bonding,

electrostatic interactions, and Van der Waals forces. Δ

S

reflects a change in the degree of order of the system

and is related to hydrophobic interactions and is the

thermodynamic property that describes the way the

molecules are distributed in a system (PIRES et al.,

2009; BOU-ABDALLAH & TERPSTRA, 2012).

Figure 1 is a graphical representation of

energy (μcal) as a function of time (s) after 18 titrations

by ITC. At the beginning of the titration, the energy

absorbed is greater due to interactions. Over time, the

rate of energy decreases until complete saturation of

binding sites is achieved. From each peak obtained in

the titration, a function chart of molar ratio can also

be constructed, which allows the variation of free

energy (Δ

G

), equilibrium binding constant (

K

), and the

stoichiometry (

n

) of reaction to be calculated. Greater

slopes of the curve represent higher binding affinities

(

K

) (PIRES et al., 2009; CERVANTES et al., 2011).

Instrument consists of two identical

cells. One is a reference, which contains only buffer

solution, and the other contains the macromolecules

solutions. Cells are made of material that conduct heat

exceptionally well and are thermally stable, with a

constant temperature variation of approximately 10

_K

(PIRES et al., 2009). Small aliquots of the titrant are

injected through a syringe into the sample cell. When the

reaction occurs, there is release (exothermic reaction)

or absorption (endothermic reaction) of energy that the

calorimeter detects (HEERKLOTZ & SEELIG, 2000).

Number, volume, and time of injections, as well as the

concentration of the samples, the cell temperature, and

rotation speed must be properly adjusted. Furthermore,

it is important that a control experiment is carried

containing only buffer solution to correct undesired

thermal effects that are related to the energy changes

during dilution and mixing (PIRES et al., 2009;

BOU-ABDALLAH & TERPSTRA, 2012).

Most ITC calorimeters use the power

compensation method that reduces temperature in

the sample cell if the reactions are exothermic, and

increases temperature if they are endothermic. Energy

absorbed or released during the titration will be directly

proportional to the interactions (BOU-ABDALLAH &

TERPSTRA, 2012). The most modern equipment are

called “nanocalorimeters” and can precisely measure

very small energy changes (<0.2mJ) and maintain a

baseline of ± 0.1mW with a temperature stability of

±0.0001°C (GAISFORD & BRUCKTON, 2001;

BOU-ABDALLAH & TERPSTRA, 2012).

Several studies have demonstrated

that ITC used in the study of macromolecular

interactions is both noninvasive and generates

a set of thermodynamic parameters. GUZEY &

MCCLEMENTS (2006), showed an exothermic

interaction between chitosan and β-LG in the pH

range in where the polymers have opposite charge.

The interaction was more exothermic at pH 6.0, with

a molar ratio of about one β-LG molecule to six

chitosan molecules.

HARNSILAWAT et al. (2006)

characterized of β-LG-sodium alginate interactions

and verify that the enthalpy change was highly

dependent on solution pH. Similarly, the interactions

between Chitosan and WPI by electrostatic bonds

were dependent not only on the pH but also the ionic

strength, and molar ratio (BASTOS et al., 2010).

In another study, the interaction between

β-LG and gum arabic in the presence of an

antioxidant were identified. The study reported a

two-step interaction, an exothermic two-step that was mostly

controlled by favorable enthalpy due to electrostatic

interactions, and a second endothermic step that was

driven by entropy, likely due to the release of linked

water molecules. In addition, this study evaluated

the influence of temperature and concluded that

the contribution of enthalpy or entropy were highly

dependent on temperature (ABERKANE et al., 2012).

ITC has been used to study the

thermodynamic of complex of coacervates used

in developing new products and encapsulation of

bioactive compounds, such as omega-3 fatty acids

and vitamin D

(WATER et al., 2014; DONG et al.,

2015; DIARRASSOUBA et al., 2015; ERATTE

et al., 2015). HOSSEINI et al. (2013) submitted

the κ-carrageenan biopolymer (KC) and β-LG on

ultrasound, and reported that KC-BLG interactions

were exothermic with negative and favorable enthalpy

and negative and unfavorable entropy. A significant

reduction in the affinity constant of the formation

of complex coacervates suggested a conformational

change. Interactions between lactoferrin and β-LG

isoforms were also identified. In this case, ITC

revealed an exothermic interaction with contributions

from both enthalpy and entropy contributions. The

study also reported that the interaction involved at

least two steps requiring two independent binding

sites (TAVARES et al., 2015).

The simultaneous use of the two

calorimetric techniques was performed by WATER

et al. (2014). The authors evaluated the formation

of complex coacervates between hyaluronic acid

and lysozyme. They reported using ITC, that the

interaction was driven entropically, characterized by

a slightly favorable binding enthalpy and a highly

favorable entropic contribution. Nonetheless, DSC

showed a reduction of

T

of about 1°C after the

complex was formed. Thus, formation of the complex

did not affect the secondary structure of the protein

and did not negatively impact its thermal stability.

CONCLUSION

Proteins and polysaccharides are

part of most food matrices. Understanding their

thermodynamic behavior and interactions with

other macromolecules is essential for food

applications. Calorimetric techniques provided

information regarding these interactions through

the thermal energy that is produced or consumed

during interactions. We have presented examples

of the efficiency and applicability of calorimetry in

studies of the interactions between macromolecules,

and highlighted the importance of thermodynamic

parameters in the interpretation of the results

obtained. Simultaneous use of DSC and ITC

are important tools that can provide a better

understanding of the macromolecular interactions

that occur during the processing and storage of food.

ACKNOWLEDGEMENTS

The authors thank Conselho Nacional de

Desenvolvimento Científico e Tecnológico (CNPq) and Fundação

de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for

the financial support.

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